Air pollution is a major worldwide concern, and exposure to particulate matter (PM) can increase the risks of pulmonary diseases. Normal human bronchial epithelial cells were applied to clarify the role of ultrafine PM (UFPM) in the pathogenesis of pulmonary toxic effects with realistic alveolar deposition doses. The UFPM used in this research originated from vehicular emissions and coal combustion. UFPM exposure of up to 72 h was found to induce significant time- and concentration-dependent decreases in cell viability. Exposure to UFPM increased reactive oxygen species (ROS) accumulation through heme oxygenase-1 (HO-1) inhibition and induced massive oxidative stress that increased the interleukin-8 (IL-8) expression. UFPM also reduced the pulmonary trans-epithelial electrical resistance through the depletion of zonula occludens (ZO) proteins. Finally, UFPM decreased the α1-antitrypsin (A1AT) expression, which implies high risk of chronic obstructive pulmonary disease (COPD). The evidence demonstrates that exposure to UFPM, even at very low concentrations, may affect the functions of the respiratory system.
Research Article
Pulmonary Toxicity of Realistic Alveolar Deposition Concentrations of Ultrafine Particulate Matters in Human Normal Bronchial Epithelial Cell
https://doi.org/10.21203/rs.3.rs-314374/v1
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Ultrafine Particulate Matter
BEAS-2B
Chronic Obstructive Pulmonary Disease
Oxidative stress
Inflammation
Particulate matter (PM) has become the subject of increasing attention for governmental, private and environmental organisations. The World Health Organization (WHO) pointed out that PM can harm the respiratory tract, causes acute respiratory tract infection and COPD and has a strong correlation with cardiovascular diseases and cancer. PM generally refers to particles or droplets suspended in the air and can be further divided based on the aerodynamic diameter into PM10, PM2.5, PM1.0 and PM0.1 (Chen et al. 2019). Both long- and short-term exposure to ambient PM have been proven to negatively affect human health. The PM source, components and size all significantly affect the toxicity. Tao et al. (Tao et al. 2014) focused on the effects of the PM concentration and size on the number of hospital admissions for respiratory and cardiovascular disorders; increasing the PM concentration by 10 µg/m3 increased the hospitalisation risk by 0.052% for PM10, 0.604% for PM2.5 and 0.652% for PM1.0. Kim et al. (Kim et al. 2015) revealed that increasing the suspended PM concentration by 10 µg/m3 resulted in a total death risk of 0.6% and 0.8% for PM10 and PM2.5, respectively. Recent research has indicated that the toxicity of atmospheric PM increases with decreasing particle size. Increasing the PM2.5 concentration by 10 µg/m3 was found to increase the total mortality by 15%, but increasing the PM10 concentration by the same amount only increased the total mortality by 0.74% (Ma et al. 2017, Martinelli et al. 2013, Velali et al. 2016). Other studies have confirmed that very small PM (<1 µm) is a key risk factor for human lungs. Hence, the effect of smaller PM sizes on human toxicity should be elucidated.
Toxicology studies have revealed that PM has several harmful cytological mechanisms, such as oxidative stress, inflammation, cytotoxicity, and DNA damage (Silva et al. 2018, Yan et al. 2017). Human cells stimulated by suspended PM may produce ROS and reactive nitrogen species. An excessive amount of highly reactive molecules may overwhelm the body’s defence, which can disrupt the equilibrium between the oxidative and antioxidative systems and lead to cell damage (Langen et al. 2003). Previous study has demonstrated that ROS can inflict oxidative damage in DNA, lipids and proteins to cause lung diseases (Reis et al. 2018). Suspended PM may accumulate on the surfaces of the bronchioles and alveoli of the lungs. As the lungs’ defence mechanisms remove inhalable particles, the macrophages, epithelial cells and other related cells generate pro-inflammatory factors that induce in situ and systematic inflammatory reactions and oxidative stress (Feng et al. 2016, Miyata &van Eeden 2011). After acute PM exposure, such particles may stimulate macrophages to release inflammatory factors, chemotactic factors and interferon-γ (Martinelli et al. 2013, Tedgui &Mallat 2006, Wu et al. 2017). Among these, IL proteins are essential inflammatory factors that have long been associated with development of COPD. During lung inflammation, neutrophil leukocytes and macrophages in the lungs secrete a large quantity of elastase that can damage the lung structure (Yuxiang et al. 2006). According to Wu et al. (Wu et al. 2017), PM can cause lung inflammation in COPD patients and increase IL expressions through the NF-κB pathway. Other studies have discovered that inflammatory factors released by pulmonary epithelial tissue can not only induce a systematic inflammatory reaction but also reach other parts of the body via the circulatory system to induce local inflammatory reactions, especially within the circulatory system itself (Tedgui &Mallat 2006).
COPD refers to chronic inflammation of the respiratory tract that can obstruct the airflow (Ritchie &Wedzicha 2020). Globally, COPD will become the third most common disease in 2020 (Chen et al. 2016). In Taiwan, COPD is the fastest rising causes of death (Chen et al. 2016). Lung obstruction is induced by oxidative stress, disrupted regulation of lung proteases and inflammatory reactions. Several studies have indicated that the imbalance between proteases and anti-proteases plays a crucial role in COPD. A1AT is a critical protease inhibitor in the body. Therefore, A1AT has frequently been associated with COPD morbidity and has even been suggested to be a biomarker of COPD (Heijink et al. 2012). Many papers have pointed out that a deficiency of A1AT protein increases the risk of COPD and can indirectly cause liver and skin diseases. A1AT can bind to and inhibit many proteases to prevent their hyperactivity. In the lungs, A1AT enters the alveoli to inhibit proteases and functions as a protective barrier against damage to the alveolar wall (Brode et al. 2012, McCarthy et al. 2016, Yuxiang et al. 2006). PM-induced ROS accumulation could lead to A1AT deficiency, and thus disrupt the pulmonary mucosal barrier integrity (Huang et al. 2014, Zuo et al. 2016).
Normal human bronchial epithelial cells (BEAS-2B cells) were applied in this research to investigate the pulmonary toxicity induced by ultrafine PM (UFPM: PM0.1–0.056) collected in Yunlin County, Taiwan. The results of this research should elucidate the role and contribution of UFPM to PM-induced lung disease.
Chemical reagents
Potassium chloride, sodium chloride, sodium phosphate, potassium phosphate, thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Sigma Co. (MO, USA). Phosphate-buffered saline, trypsin inhibitor, trypan blue, RIPA buffer and LHC-9 medium were obtained from Thermo Fisher Scientific (MA, USA). Trypsin (0.05% and 0.25%) was purchased from Corning (VA, USA).
Sampling, characterization and preparation of UFPM
A model-110R micro-orifice uniform deposited impactor (MOUDI II™) (MN, USA) was used to collect UFPM samples in Yunlin County of western Taiwan. Yunlin County is located near one of the highest-polluting industry and have a poor air quality (Hsu et al. 2017). Samples were taken every 24 h from December 2017 to February 2018. All filters were conditioned (humidity of 45±5% and temperature of 25±2°C) and then weighed. The UFPM was classified as PM0.18–0.056 (i.e. aerodynamic diameter of 0.18–0.056 mm). The metallic elements and polycyclic aromatic hydrocarbons (PAHs) of the UFPM were determined via inductively coupled plasma–mass spectrometry (Finnigan, Germany) and gas chromatography–mass spectrometry (Finnigan, USA). The UFPM samples were mixed with deionised water and distilled in an ultrasonic bath. Finally, the UFPM samples were freeze-dried and then stored (−20°C).
Cell culture
BEAS-2B cells were cultured in LHC-9 and maintained in CO2 incubator. The LHC-9 was replaced twice weekly. The BEAS-2B cell line was purchased from ATCC. This research does not use any human tissue or animal. According to Health Research Authority, research using cell lines does not require ethical review.
MTT assay
BEAS-2B cells (8 × 103 cells/well) were co-treated with UFPM (3–36 µg/ml) for up to 72 h. To measure the cytotoxicity, an MTT assay was performed following the manufacturer’s instructions. The intensity of samples was determined with a multilabel reader (Perkin Elmer, MA, USA) at a wavelength of 490 nm. UFPM samples were removed before the absorbance was measured to avoid interference. The BEAS-2B cell viability was expressed as a percentage of non-treated group.
DCFH-DA assay
BEAS-2B cells were co-treated with UFPM (3–36 µg/ml) for 48 h. DCFH-DA assay was performed to determine the oxidative stress. The cells were seeded in 24-well cell culture plates (5 × 104 cells/well) overnight. Culture medium containing DCFH-DA and UFPM were added and incubated for up to 48 h. H2O2 was used as a positive control. DCF fluorescence images were taken with fluorescence microscopy (Nikon, Tokyo, Japan), and the fluorescence intensity was quantified by using ImageJ software.
Enzyme-linked immunosorbent assay
For the ELISA, BEAS-2B cells were seeded in 96-well cell culture plates (8 × 103 cells/well) overnight. After exposure to UFPM (12 and 36 µg/ml) for 48 h, the cell culture medium was collected to determine the expressions of A1AT, IL-6 and IL-8 following the manufacturer’s instructions (R&D Systems, Minneapolis, MN). A multilabel reader (Perkin Elmer, MA, USA) was used to measure the absorbance at 450 and 540 nm. UFPM was removed before the absorbance was measured to avoid interference.
Western blotting
After exposure to UFPM (12 and 36 µg/ml) for 48 h, BEAS-2B cells were washed with PBS and lysed in protein extraction buffer. The lysate was centrifuged and then the supernatant was collected for protein expression analysis. The protein concentration was determined by Bio-Rad assay kit (CA, USA). Protein (40 µg/lane) was loaded and separated by SDS-PAGE gradient gel (6%–12%). The samples were then electrophoresed and electroblotted onto a PVDF membrane. The PVDF membranes were blocked in TBS with 5% nonfat milk. The expressions of the ZO and HO-1 proteins were detected with 1:1000 antibodies. Horseradish-peroxidase-coupled anti-rabbit or anti-mouse antibodies (1:1000) were used as the secondary antibody. The chemiluminescence signals were quantitatively analysed using an imaging system (MultiGel-21, TOPBIO, Taiwan). The anti-β-actin antibody-probed membranes were used as the loading control.
Trans-epithelial electric resistance (TEER)
After the BEAS-2B cells reached 100% confluence on the Transwell insert, they were treated with UFPM (12 and 36 µg/ml). To determine the cell barrier integrity, the Millicell® ERS-2 system (Merck Ltd., Taiwan) was used to measure the TEER after exposure to UFPM (24–72 h). The barrier resistance (Ω/cm2) was determined by subtracting the resistance of the blank insert from the TEER.
Statistical analysis
The significance of differences in different groups was determined by one-way analysis of variance followed by Dunnett’s test. The statistical significance was expressed as *p < 0.05.
Environmental and human alveolar deposition concentrations of UFPM
In this research, the sampling region (23°70′28.35″ N, 120°42′88.20″ E) is surrounded by schools, residential buildings and non-residential buildings such as office spaces, retail shops and restaurants (Figure 1). The daily mean UFPM concentration was 12.1 ± 6.03 µg/m3 (7.18–23.4 µg/m3) on the sampling period (Table S1), which is less than the limit specified by WHO for PM2.5 (25 µg/m3) (Organization 2005). The particle size is a key factor that influences PM toxicity, so UFPM may have health impacts even at low concentrations. The daily UFPM alveolar deposition dose was determined as previously reported with modifications (Erdely et al. 2013).
where VE is the respiratory rate. The alveolar deposition efficiency of UFPM was determined by the International Commission on Radiological Protection Deposition Model (14.2%). Therefore, the daily UFPM alveolar deposition dose was estimated as 11.1 ± 5.54 µg/day. The alveolar deposition doses for 8, 48 and 72 h of UFPM exposure were calculated to be 3.70 ± 1.85, 22.2 ± 4.85 and 33.3 ± 7.28 µg/day, respectively (Table S1). Based on the above data, the UFPM concentrations for the cytotoxic tests were set to 3, 12, 24 and 36 µg/ml.
Chemical Composition of UFPM
PM toxicity is mainly related to its chemical composition. The UFPM samples were characterised. In total, 19 PAHs were measured, and the major species were benzo[ghi]perylene (956.0 pg/m3), indeno[123-cd]pyrene (692.6 pg/m3), benzo[b]fluoranthene (395.1 pg/m3), benzo[k]fluoranthene (263.5 pg/m3), benzo[e]pyrene (186.9 pg/m3), benzo[a]pyrene (174.4 pg/m3) and dibenz(a,h)anthracene (Table S2). PAH diagnostic ratios can be used to assess their pollution emission sources and age. In Table 3, the ratio of Fluorene/(Pyrene+Fluorene), Anthracene/(Phenanthrene+ Anthracene), Fluoranthene/(Pyrene+Fluoranthene), Benzo(a)anthracene/(Chrysene+Benzo(a)anthracene), Benzo(a)pyrene/(Benzo(e)pyrene+ Benzo(a)pyrene), Indeno(1,2,3-c,d)pyrene/( Benzo(g,h,i)perylene+Indeno(1,2,3-c,d)pyrene) and 2-Methylnaphthalene/Phenanthrene were 0.11, 0.15, 0.53, 0.36, 0.48, 0.42 and 0.32 (Table 1). These results suggested that the UFPM samples primarily came from vehicular emissions and coal combustion (Tobiszewski &Namiesnik 2012, Zhao et al. 2011). The dominant metallic elements of the UFPM samples were Sr (595.4 pg/m3), Fe (233.3 pg/m3), Al (1.784 pg/m3), Zn (0.662 pg/m3) and Ni (0.389 pg/m3) (Table S3), which have crustal and anthropogenic sources. Human activities including manufacturing and the usage of ceramics, glass, fireworks products and phosphate fertilisers release Sr into the environment (Höllriegl &München 2011). Fe and Zn are associated with vehicle emissions, whereas Al is associated with dust (Liu et al. 2014, Lu et al. 2005). Ni may come from fuels (Genchi et al. 2020). Exposure to Al and Ni is proven to cause COPD-like effects (Li et al. 2017) and pulmonary cancer (Genchi et al. 2020). Moreover, high Fe serum concentration have been detected in COPD patients (Asker et al. 2018). These results indicate that residents in the sampling area may potentially at risk of developing respiratory diseases.
Table 1. Diagnostic rations of PAHs
PAHs ratio |
Value |
|
Fluorene/(Pyrene+Fluorene) |
0.11 |
|
Anthracene/(Phenanthrene+Anthracene) |
0.15 |
|
Fluoranthene/(Pyrene+Fluoranthene) |
0.53 |
|
Benzo(a)anthracene/(Chrysene+Benzo(a)anthracene) |
0.36 |
|
Benzo(a)pyrene/(Benzo(e)pyrene+Benzo(a)pyrene) |
0.48 |
|
Indeno(1,2,3-c,d)pyrene/(Benzo(g,h,i)perylene+Indeno(1,2,3-c,d)pyrene) |
0.42 |
|
2-Methylnaphthalene/ Phenanthrene |
0.32 |
Cytotoxic and oxidative effects induced by UFPM
The lung epithelial barrier is the frontline of defence against inhaled extraneous materials, and damage to this barrier is an early marker of respiratory diseases (Frey et al. 2020, Gao et al. 2015). BEAS-2B cells were treated with UFPM at realistic inhaled concentrations (3, 12, 24 and 36 µg/ml) for up to 72 h, and the cytotoxicity was determined. After 24 and 48 h of exposure, significant cell reduction was only observed with 36 mg/ml of UFPM exposure (~80%–90% of control; Figure 2). However, significant cytotoxicity was observed after 72 h of exposure, even at the lowest concentration of 3 mg/ml (~65%–48% of control; Figure 2).
Pulmonary oxidative stress has a pathogenic role in lung diseases (Ornatowski et al. 2020, Rahman 2005). To detect the UFPM-induced oxidative response, BEAS-2B cells were treated with UFPM for 48 h at non-cytotoxic (12 µg/ml) and cytotoxic (36 µg/ml) concentrations, and the ROS formation and antioxidative enzyme (HO-1) expressions were determined. ROS formation significantly increased in both UFPM-treated groups (Figures 3A and 3B). By contrast, the HO-1 activity significantly decreased in both UFPM-treated groups (Figures 3C and 3D). These results indicate that UFPM induces a dose-dependent oxidative response in BEAS-2B cells. UFPM may induce ROS accumulation by overwhelming the antioxidant enzyme capacity (Ambreen et al. 2020).
Inflammatory effects induced by UFPM
The oxidative stress response has been associated with the inflammatory response (Han et al. 2009, Yang et al. 2020). BEAS-2B cells were exposed to UFPM (12 or 36 µg/ml) for 48 h, and the expressions of pro-inflammatory cytokines were determined. Neither UFPM-treated group had significant increases in IL-6 protein expression (Figure 4A). Although the IL-8 protein expression decreased as the UFPM concentration increased, exposure to UFPM increased the IL-8 protein expression (Figure 4B). The oxidative cytotoxic effect induced by a high UFPM concentration may contribute to inflammation. Generally, UFPM was found to exhibit cytotoxic effects for ROS accumulation and inflammation. COPD has been associated with an increased oxidative response (MacNee 2001, Ornatowski et al. 2020). Thus, inhalation exposure to UFPM may increase the risk of pulmonary diseases.
Epithelial barrier dysfunction induced by UFPM
Pulmonary epithelial barrier dysfunction can increase susceptibility to lung injury (Wang et al. 2020, You et al. 2012). A continuous epithelial cellular barrier is organized through tight junctions, such as zonula occludens connecting adjacent cells to prevent invaders from diffusing through to internal pulmonary tissue (Li et al. 2021). BEAS-2B cells were treated with 12 or 36 µg/ml of UFPM for 48 h to examine the epithelial barrier disruption induced by UFPM. The TEER significantly decreased for the UFPM-treated epithelial barrier (Figure 5A). Previous research has shown that impaired pulmonary barrier functionality is related to the downregulation of ZO-1 (Li et al. 2021, You et al. 2012). In the ELISA results, the UFPM-treated cells showed significant reductions in ZO-1 and ZO-2 activity (Figures 5B-D). UFPM induces dysfunction of the epithelial barrier through tight junction disruption; however, barrier dysfunction alone may be insufficient to promote pulmonary diseases.
Potential contributions of UFPM to the pathogenesis of COPD
Long-term exposure to ambient PM is associated with the increased prevalence and incidence of COPD (Atkinson et al. 2015, Han et al. 2020). To elucidate the positive contribution of UFPM to COPD pathogenesis, BEAS-2B cells were treated with 12 or 36 µg/ml of UFPM for 48 h. The main function of A1AT is to balance the neutrophil elastase and protect the lungs from protease-induced excess destruction of pulmonary tissue (Meseeha &Attia 2020). Both UFPM-treated groups showed significant decreases in the A1AT protein expression (Figure 6). The UFPM-induced imbalance of A1AT may increase the risk of developing COPD (Li et al. 2009, Murphy et al. 2020). Clearly, UFPM is a major contributor to PM-induced COPD.
Although PM exposure has been proven to contribute to the progression of COPD, little research has focused on clarifying the toxic potential and molecular mechanisms of UFPM. In this study, normal human pulmonary epithelial cells were used to evaluate the health impact of UFPM at environmentally realistic concentrations. The results indicated that inhalation of UFPM even at non-cytotoxic concentrations can instigate ROS accumulation, leading to inflammation and epithelial barrier dysfunction. These adverse effects can inhibit A1AT activity and contribute to the development of COPD. These findings suggest that there should be deep concerns over the respiratory health issues associated with UFPM.
Ethics approval and consent to participate: This research does not involve human participants, human data, human tissue or animal. According to Health Research Authority, research using cell lines does not require ethical review and consent to participate.
Consent to Publish: I, Chia-Hua Lin hereby declare that I participated in the study and in the development of the manuscript titled “Pulmonary toxicity of realistic alveolar deposition concentrations of ultrafine particulate matters in human normal bronchial epithelial cell”. I have read the final version and give my consent for the article to be published in Environmental Science and Pollution Research.
Authors Contributions: Chia-Hua Lin was responsible for research design, experimental performance and drafting the manuscript. Shih-Chun Candice Lung made contribution to sample collection and experimental technical support. Yi-Chun Chen and Lung-Chun Wang made contribution to analyse the data and the technical assistance. Yi-Chun Chen contributed significantly to the manuscript preparation.
Funding: This work was supported by the Ministry of Science and Technology, Taiwan (106-2314-B-150-001 and 107-2221-E-150-004-MY3).
Competing Interests: To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise.
Availability of data and materials: The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
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